?Control of cap-independent translation by N6-methyladenosine and FTO? 1 R01 CA186702-01 PI: Jaffrey, Samie R. SUMMARY The ability of cancer cells to dynamically change their protein composition is a major mechanism by which they respond to diverse types of cellular stress, such as DNA damage and chemotherapeutics. In many cases, cellular stresses activate novel modes of translation that enable the expression of specific proteins that enhance the cancer phenotype. Cancer-specific translation controls the expression of various proteins with roles in cell survival and adaptation to stress. Novel sequence elements and motifs in the 5?UTR of mRNAs are thought to have key roles in conferring the ability of transcripts to exhibit cancer-specific translation. However, their function remains mysterious because they typically lack sequence conservation. Moreover, it has not been possible to recapitulate their effects using in vitro assays that reconstitute translation initiation. In this proposal, we propose to examine the function of a recently described mRNA base modification, N[6]-methyladenosine (m6A), and determine how it affects translation of mRNAs in cancer cells. Furthermore, we previously showed that the cancer risk gene, fat mass and obesity associated gene (FTO), selectively demethylates m6A in mRNA, suggesting that it may have a role in regulating mRNA fate or translation in cells. In order to significantly advance our understanding of this novel pathway that regulates protein translation, our goals are: (1) To develop SIMPL-Seq: The first method to map m6A at single-nucleotide resolution. Although m6A is known to occur in ~8,000 different mRNAs, it is not currently possible to determine its precise location in mRNAs. This is necessary for mutating these residues and establishing the function of m6A in translation and other processes. In this aim we use a novel chemical biology approach to map, for the first time, m6A residues throughout the transcriptome of diverse cancer cell lines. (2) To determine how m6A regulates translation of mRNA. To understand the mechanism by which m6A influences translation, this aim will address: (1) the sequence and structural features of m6A-containing sequence motifs that enable it to affect each type of translation initiation seen in cancer cells; (2) the m6A-binding proteins that influence ribosome recruitment to mRNA; and (3) the role of m6A in promoting translation in living cells. These experiments use both in vitro reconstitution and cell-based experiments to provide an understanding of translational regulation mediated by m6A. (3) To test the role of FTO as a regulator of translation. In this aim, we will determine if FTO is a physiologic regulator of translation. To do this, we will use ribosome profiling to identify transcripts that are translated through diverse modes of translation initiation, and determine if these are affected by FTO knockdown or overexpression. These experiments will test whether FTO functions to ?switch off? translation that is induced by m6A. Together, the experiments in these three aims seek to resolve the long-standing mystery regarding the regulation of cancer-specific translation pathways in cells. This proposal describes a functional role for m6A and tests the idea that FTO is a regulator of translation pathways in cells. These studies will redefine our basic understanding of pathways controlling translation in cells and provide a basis for further exploration of the role of m6A and FTO in cancer and other diseases.